Myc proteins are transcription factors crucial for cell proliferation. They have a C-terminal domain that mediates Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: Trp77-Glu86 and Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with the phosphodegron, have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and resulted in no significant structural changes outside the phosphodegron. When the phosphodegron was doubly phosphorylated, N-myc formed a robust interaction with the Fbxw7-Skp1 complex, but mapping the interaction by NMR suggests a more extensive interface. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

中文翻译：

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通过溶液核磁共振波谱探索 N-myc 反式激活结构域的动力学和相互作用。


Myc 蛋白是细胞增殖至关重要的转录因子。它们有一个介导 Max 和 DNA 结合的 C 端结构域，以及一个以反式激活结构域 （TAD） 为终点的 N 端无序区域。TAD 参与许多蛋白质-蛋白质相互作用，特别是与通过泛素-蛋白酶体系统促进稳定性 （Aurora-A） 或降解 （ERK1、GSK3） 的激酶相互作用。在完成主干分配后，我们使用核磁共振 （NMR） 波谱研究了 N-myc TAD 的结构、动力学和相互作用。化学位移分析显示，N-myc 有两个具有明显螺旋倾向的区域：Trp77-Glu86 和 Ala122-Glu132。这些区域也比 TAD 的其余部分具有更受限的 ps-ns 运动，并且与磷酸化 （phosphodegron） 一起具有相对较高的横向 （R2） 15N 弛豫速率，表明时间尺度动力学和/或化学交换较慢。总的来说，这些特征表明整个 TAD 中结构和相互作用的差异倾向，无论是内部的还是与结合伴侣的。关于 N-myc 和 Aurora-A 之间相互作用的解决方案研究揭示了一个以前未表征的结合位点。使用 NMR 表征 ERK1 和 GSK3 对 N-myc 连续磷酸化的特异性和动力学，并且不导致磷酸化体外的显着结构变化。当磷酸化 degron 被双重磷酸化时，N-myc 与 Fbxw7-Skp1 复合物形成稳健的相互作用，但通过 NMR 绘制相互作用图表明存在更广泛的界面。 我们的研究提供了对 N-myc TAD 动力学的基础见解和主干分配，这将为未来关于该关键癌蛋白的结构、动力学、相互作用和调节翻译后修饰的工作奠定基础。